The long term goal of this project is to understand how a signal originating at the cell periphery triggers a series of events culminating in the synthesis and assembly of a cellular organelle. Methods have been developed for deciliating starved Tetrahymena, resulting in a reproducible series of events including the rapid induction of the synthesis of a deciliation induced protein (referred to as DIP) followed by synthesis of tubulin and regeneration of cilia. DIP and tubulin synthesis are induced at least 15-fold and can represent 15% (DIP) and 7.5% (tubulin) of the proteins synthesized at appropriate periods of regeneration. Many of the methods of modern molecular and cellular biology will be utilized in attempts to pin-point the steps at which regulation occurs. Recombinant DNA clones containing tubulin and DIP mRNA sequences will be prepared and used to study the relative contributions of transcriptional and post-transcriptional events in the regulation of the abundance of tubulin and DIP mRNAs. The relationships between mRNA abundance and mRNA translation also will be examined. The structures of tubulin and DIP primary transcripts and the mRNA molecules themselves will be analyzed to determine whether changes in gene expression are related to changes in hnRNA or RNA molecules. The organization of tubulin and DIP sequences in the genome and the structural changes in chromatin (if any) which accompany changes in the expression of these genes will also be studied. A detailed analysis of the (ciliary and non-ciliary, nuclear and cytoplasmic) proteins which are synthesized and accumulated during regeneration will be undertaken in a search for molecules whose properties suggest they play an important role in regulating cilia regeneration. When we have a detailed description of the types, amounts and cellular locations of RNAs and proteins, we shall isolate and analyze conditional mutants in cilia regeneration in order to probe the mechanisms regulating gene expression and organelle biogenesis in greater detail.